The dorsoventral axis of the Drosophila embryo is established by ventral activation of the Toll signaling pathway, resulting in the graded nuclear uptake of the Dorsal transcription factor. Pathway activation relies on a ventral cue synthesized during oogenesis through the action of Pipe, a putative heparan sulfate 2-O- sulfotransferase, on an unknown carbohydrate substrate. In the embryo, the ligand for the receptor Toll is generated by a proteolytic processing reaction involving 4 members of a serine protease cascade, Nude1, Gastrulation Defective (GD), Snake and Easter. Published work has shown that early cascade events occur independently of pipe activity and are not ventrally restricted, while new data shows that activation of the Easter protease requires pipe and is probably ventrally restricted. These observations establish that spatial control occurs within the protease cascade. To investigate the mechanism by which pipe regulates the cascade, three aims are proposed to test the hypothesis that glycosaminoglycans (GAGs) modified by Pipe serve as cofactors promoting the ventral activity of GD and/or Snake. These include: (1) determining where spatial regulation occurs in the protease cascade in vivo, by Western blotting and localization of active proteases; (2) characterizing the functional consequences of GAG interactions with GD and Snake in vitro using purified proteases, and determining whether the specificity of these interactions is consistent with that expected for Pipe-modified GAGs; and (3) identifying GAGs and proteoglycans involved in protease regulation by pipe in vivo, using novel selection strategies to isolate species structurally altered in the absence of pipe. This work is relevant to understanding the regulation of protease cascades, such as those involved in the innate immune response, hemostasis, wound healing, and synaptic plasticity. Additionally, knowledge of how glycosaminoglycan microheterogeneity contributes to patterning is important to understanding the many emerging roles of heparan sulfate proteoglycans in vertebrate and invertebrate development, where, for example, a knock-out of a mouse gene related to pipe results in renal agenesis and skeletal and eye defects.